Nature of PEVK-titin elasticity in skeletal muscle

Abstract

A unique sequence within the giant titin molecule, the PEVK domain, has been suggested to greatly contribute to passive force development of relaxed skeletal muscle during stretch. To explore the nature of PEVK elasticity, we used titin-specific antibodies to stain both ends of the PEVK region in rat psoas myofibrils and determined the region's force-extension relation by combining immunofluorescence and immunoelectron microscopy with isolated myofibril mechanics. We then tried to fit the results with recent models of polymer elasticity. The PEVK segment elongated substantially at sarcomere lengths above 2.4 micro(m) and reached its estimated contour length at approximately 3.5 micro(m). In immunofluorescently labeled sarcomeres stretched and released repeatedly above 3 micro(m), reversible PEVK lengthening could be readily visualized. At extensions near the contour length, the average force per titin molecule was calculated to be approximately 45 pN. Attempts to fit the force-extension curve of the PEVK segment with a standard wormlike chain model of entropic elasticity were successful only for low to moderate extensions. In contrast, the experimental data also could be correctly fitted at high extensions with a modified wormlike chain model that incorporates enthalpic elasticity. Enthalpic contributions are likely to arise from electrostatic stiffening, as evidenced by the ionic-strength dependency of titin-based myofibril stiffness; at high stretch, hydrophobic effects also might become relevant. Thus, at physiological muscle lengths, the PEVK region does not function as a pure entropic spring. Rather, PEVK elasticity may have both entropic and enthalpic origins characterizable by a polymer persistence length and a stretch modulus.

Figure 1

I-band titin domain architecture showing the splice variant likely to be found in psoas muscle (1, 13). The epitope positions of the two antibody types used in this study, N2-A and I20/I22, are indicated. Note that not the entire I-band titin is elastic; a 100 nm-long segment at the Z-disc end is functionally stiff. Ig, Ig-like; FN3, fibronectin type-3-like.

Figure 2

Examples of immunostainings with titin antibodies flanking the PEVK region. (A and B) Immunofluorescence images of single psoas myofibrils stretched to different SLs (indicated on the right) and labeled with either the N2-A or the I20/I22 titin antibody (A) or both antibody types together (B). As secondary antibody, Cy-3-conjugated IgG was used. In B, the lower two images show the same myofibril both at high and moderate stretch; the spacing between two close fluorescent stripes indicates the length of the PEVK region. pc, phase-contrast images. (Scale bar, 5 μm.) (C) Immunoelectron micrographs of stretched psoas muscle sarcomeres stained with N2-A or I20/I22. The nanogold particles indicate the respective epitope positions (arrows). (Scale bar, 0.5 μm.)

Figure 3

Extension behavior of the PEVK segment. (A) Summary of results of immunolabeling experiments. Immunoelectron microscopy data points for the Z-line center to epitope spacing are shown for both N2-A (□) and I20/I22 (•). The larger shaded circles (and triangles) and error bars indicate the mean distances from the Z-disc center and SD for N2-A (and I20/I22, respectively), measured by immunofluorescence microscopy (IF). Curve fitting parameters (electron microscopy data only) also are shown. (B) Extension of the PEVK region vs. SL. The left axis indicates the region’s end-to-end length, the right axis fractional extension (relative to the segment’s assumed contour length of 476 nm).

Figure 4

Force-length relations of titin. (A) Force per single titin molecule at different SLs, extrapolated from the steady-state passive tension vs. SL curve of isolated rat psoas myofibrils, shown in the Inset. The curve parameters also are indicated. (B) Force per titin vs. fractional extension of the PEVK region. Open circles and error bars indicate the mean force and SD of incrementally summarized data points and the thick shaded line the calculated fit to the experimental data, both deduced from A and Fig. 3B. The dotted black line represents the WLC model fit according to Eq. 1 (entropic elasticity), the continuous black line the modified WLC fit according to Eq. 2 (entropic-enthalpic elasticity). (Inset) For comparison, force vs. fractional extension (thicker shaded line) and fit according to the standard WLC model with A = 21 nm (thinner black line), calculated for the N-terminal poly-Ig segment of rat psoas titin (20).

Figure 5

Titin-based stiffness of actin-extracted rat myofibrils at different ionic strengths. (Inset) Representative force oscillations of psoas myofibrils (raw data filtered with Butterworth bandpass filter) in response to 20-Hz oscillatory motor movement. The magnitude of force oscillation—a measure of myofibril stiffness—increases progressively as IS is lowered from 170 to 50 and 20 mM. The effect was reversible on returning to 170 mM. The main figure shows the average IS-dependent stiffness change (relative to the stiffness at normal IS) of actin-extracted psoas (ps; n = 6) and right ventricular (rv; n = 9) myofibrils.